Genetically engineered animals offer the potential for tremendous advances in the production of valuable pharmaceutical products from the cells of such animals. However, the production of genetically modified animals involves significant technical hurdles that have only been overcome for a few species. The ability to incorporate genetic modifications into the permanent DNA of a species requires several distinct technologies that must be developed for each genetically engineered species. One approach to alter the genetic and physical characteristics of an animal is to use embryonic stem cells that contribute to the phenotype of an animal when injected into an embryonic form of the animal. Embryonic stem cells have the ability to contribute to the tissue of an animal born from the recipient embryo and to contribute to the genome of a transgenic organism created by breeding chimeras.
Significant expenditure of time and resources has been committed to the study and development of embryonic stem cell lines, the manipulation of the genome of the cells, and cell culture techniques that permit such engineered cells to be maintained in culture. Although many attempts have been made, the ability to sustain the pluripotency of engineered embryonic stem cells in culture has been achieved for only a few species, notably mice. For other species, the promise of genetic engineering in transgenics for protein production has been frustrated by the lack of sustainable long-term embryonic stem cell cultures.
If sustainable cultures of embryonic stem cells were readily available and susceptible to genetic engineering while maintaining pluripotency, a broad application of new technologies would be available. Because embryonic stem cells contribute to the permanent DNA of an animal, the physiological characteristics of the animal from which an embryonic stem cell was derived can be transferred to a recipient embryo by incorporating these cells into the recipient animal in an embryonic state. This offers two principal advantages: first, the phenotype of an animal from which embryonic stem cells are derived can be selectively transferred to a recipient embryo. Second, as noted above, when the embryonic stem cell cultures are particularly stable, the genome of the cells can be modified genetically to introduce genetic modifications into a recipient embryo in which the cells are introduced.
In certain cases, the embryonic stem cells can be engineered with a transgene that encodes an exogenous protein. The transgene is a genetic construct that contains DNA that acts as the blueprint for the production of a valuable protein and contains sufficient coding and regulatory elements to enable the expression of the protein in the tissue of the animal that is created from the insertion of the stem cells into a recipient embryo. In many cases, the expression of a protein is particularly valuable because the protein can be collected and isolated from the transgenic animal. However, the collection of a valuable protein from the tissues of an animal typically requires that the expression be limited to certain specific tissues that facilitate collection of the expressed protein. For example, in cows, the expression of a protein in the milk would enable the ready collection of the protein by simply collecting the milk of the cow and separating the exogenous protein. In chickens, the robust production of proteins in the white of the egg also provides an attractive vehicle for the expression of exogenous proteins. If the expression of valuable proteins could be achieved in this manner, the animal could be used as a vehicle for production of proteins that is superior to other production methods. Thus, one particularly attractive field of research and attractive area for commercial development is genetically engineered animals that express selected exogenous proteins in specific tissue that facilitate isolation and collection of the protein. The ability to produce exogenous proteins in specifically selected cells of an animal is also particularly valuable because the absence of tissue specificity simply results in the protein being expressed in all of the tissues of an animal. Under such circumstances, it is unlikely that a meaningful quantity of the protein could be separated from the animal, and furthermore the ubiquitous expression of an exogenous protein is usually very damaging to the overall health and well being of the animal.
If an embryonic stem cell culture is sufficiently stable to allow a transgene to become integrated into the genome of the embryonic stem cell, a transgene encoding tissue specific expression of a protein can be passed to a new chimeric or transgenic organism by several different techniques depending on the specific construct used as the transgene. Whole genomes can be transferred by cell hybridization, intact chromosomes by microcells, subchromosomal segments by chromosome mediated gene transfer, and DNA fragments in the kilobase range by DNA mediated gene transfer (Klobutcher, L. A. and F. H. Ruddle, Annu. Rev. Biochem., 50: 533-554, 1981). Intact chromosomes may be transferred to an embryonic stem cell by microcell-mediated chromosome transfer (MMCT) (Foumier, R. E. and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A., 74: 319-323, 1977).
As noted above, the performance of genetic modifications in embryonic stem cells to produce transgenic animals has been demonstrated in only a very few species. For mice, the separate use of homologous recombination followed by chromosome transfer to embryonic stem (ES) cells for the production of chimeric and transgenic offspring is well known. Powerful techniques of site-specific homologous recombination or gene targeting have been developed (see Thomas, K. R. and M. R. Capecchi, Cell 51: 503-512, 1987; review by Waldman, A. S., Crit. Rev. Oncol. Hematol. 12: 49-64, 1992). Insertion of cloned DNA (Jakobovits, A., Curr. Biol. 4: 761-763, 1994), and manipulation and selection of chromosome fragments by the Cre-loxP system techniques (see Smith, A. J. et al., Nat. Genet. 9: 376-385, 1995; Ramirez-Solis, R. et al., Nature 378: 720-724, 1995; U.S. Pat. Nos. 4,959,317; 6,130,364; 6,091,001; 5,985,614) are available for the manipulation and transfer of genes into murine ES cells to produce stable genetic chimeras. Many such techniques that have proved useful in mammalian systems would be available to be applied to non-mammalian embryonic stem cells if the necessary cell cultures were available and if transgenes could be designed that yielded tissue specific expression in specific tissues that facilitate isolation and collection of the exogenous protein.
The transgenes that enable tissue specific expression are complex and the genetic manipulations that are necessary to incorporate the transgenes into an embryonic cell line require extensive manipulation of the embryonic stem cells and can threaten the pluripotency of the stem cells unless the culture conditions are optimized for transgenesis. Thus, embryonic stem cell lines suitable for use in transgenesis must be both stable in culture and must maintain pluripotency when the ES cell is transfected with a genetic construct that is large and complex enough to contain all of the elements necessary for protein expression. Moreover, the genetic construct must be expressed in the ES cell to allow selection of successfully transformed cells, and the ES cell must maintain potency and the transgene must remain viable during the injection into recipient embryos and the formation of resulting animals. In the resulting animal, the transgene must be effectively expressed in specific individual tissue types in which the transgene is designed to be expressed, and, should not be expressed in other tissues such that the viability of the animal is compromised. For example, transgenes encoding DNA derived from the lymphoid elements of the immune system might be specifically targeted to be expressed in B lymphocytes of a chimeric or transgenic animal. Specifically for the expression of valuable proteins, the transgene may be designed to express protein in the oviduct such that the resulting protein is deposited in egg white. In such circumstances, the embryonic stem cell culture must allow transformation of the genome of the embryonic stem cell with a transgene containing DNA encoding an exogenous protein, the embryonic stem cell must contribute significantly to the genome of the resulting animal, and the exogenous protein expression must be tissue specific and substantially exclusive to particular tissues or tissue types. For example, to enhance the phenotype of a chimeric or transgenic animal, tissue-specific expression of an exogenous protein may be desired in the gut, liver, or pancreas. In an avian species, tissue-specific expression of exogenous proteins in the cell types of the oviduct would yield a valuable biological system for protein production.
For the production of exogenous proteins, avian biological systems offer many advantages including efficient farm cultivation, rapid growth, and economical production. Globally, chickens and turkeys are a major source of protein in the human diet. Further, the avian egg offers an ideal biological design, both for massive synthesis of a few proteins and ease of isolation and collection of protein product. However, application of the full range of mammalian transgenic techniques to avian species has been unsuccessful. Most notably, the transmission to a mature, living animal of a genetic modification encoding an exogenous protein introduced into an avian embryonic stem cell and expressed with tissue specificity has not been demonstrated.
As is described in further detail below, avian transgenesis requires avian embryonic stem cells that are stable in culture and maintain pluripotential capability, defined as exhibiting an embryonic stem cell phenotype, for an extended period of time sufficient to introduce a transgene enabling tissue specific expression of an exogenous protein to be incorporated in the genome of the ES cell. Unless the ES cell culture conditions are ideal, embryonic stem cells begin to differentiate in a short period of time and lose the ability to-contribute to the somatic tissue of a chimeric organism derived from an embryo in which the cells are injected. Thus, when differentiation occurs, cells in culture are no longer useful as pluripotential cells and also cannot be used for transgenesis. Using current avian ES cell culture techniques, the short time periods during which ES cells maintain pluripotency in culture limits their use in creating chimeras and prevents the ability to create desirable chimeric or transgenic avians, specifically those expressing exogenous proteins.
In many cases, the techniques necessary to introduce genetic modifications into embryonic stem cells, the screening of modified embryonic stem cells to select specific cell modifications in which the genetic constructs have been introduced, and the ability to manipulate the ES cells for injection into embryos to produce transgenic chickens, requires at least several weeks for all of the steps to be performed. For the embryonic stem cells to be useful in transgenesis, the pluripotential state must be maintained for the entire time period up until injection into an embryo and the ES cell must be incorporated into a recipient embryo to a degree necessary to express meaningful quantities of the exogenous protein in the resulting animal.
The specific design of the transgene also must consider the content of the DNA sequences encoding the exogenous protein, the specific tissue in which expression is targeted, the host organism in which expression occurs, and the nature of the protein to be expressed. Because proteins ordinarily expressed in vivo vary dramatically in their size, biochemical characteristics, and functionality, the transgene designed for tissue specific expression must satisfy several parameters to enable successful integration into the genome of an embryonic stem cell and to insure successful expression in the selected tissue of the host organism.